Mitochondria are dynamic, essential, double-membraned organelles that perform a myriad of tasks within cells. Unlike their bacterial ancestors, they are not discrete entities. Isolated mitochondria are transient and in communication via fusion to form both localized and widespread mitochondrial syncytia within cells. Mitochondrial division antagonizes fusion and together these events function to create a compartment that is connected, with access to mtDNA, and thus functional, yet able to be distributed to distant cellular destinations via transport on actin or microtubule networks. We are focused on understanding the molecular mechanism of mitochondrial fusion to gain insight into the roles and regulation of fusion in cells and disease. In addition to its fundamental cellular role, mitochondrial fusion also regulates intrinsic apoptosis in cells and conversely, the pro-apoptotic Bcl-2 proteins regulate mitochondrial fusion in healthy cells. The important cellular roles of fusion are underscored by the fact that mitochondrial fusion is required for embryonic development and mutations in fusion proteins cause neurodegenerative diseases and stroke. Mitochondrial fusion is unique in that it is mediated by the action of highly conserved dynamin-related proteins (DRPs). DRPs are large GTPases that, through their ability to self-assemble and hydrolyze GTP, control membrane remodeling events. Our powerful yeast in vitro assay has revealed a wealth of information regarding the fusion mechanism. We have demonstrated that DRPs mediate both membrane tethering and lipid-mixing steps in fusion at the mitochondrial outer and inner membranes. Our recent work also indicates that a non-DRP outer membrane fusion protein is required post-membrane tethering, at the lipid-mixing step of outer and inner membrane fusion. Building on our success, we have now reconstituted the analogous mammalian mitochondrial fusion reaction in vitro. Using our yeast and mammalian systems, we will determine the fundamental mechanism of mitochondrial fusion, explore the mechanistic significance of the unique features of the mammalian mitochondrial fusion machines, and determine the mechanistic basis of the regulation of mitochondrial fusion by Bcl-2 proteins. Data from the proposed experiments will provide a foundation to understand the physiological roles mitochondrial fusion plays in cells. In addition, they will directly provide information that illuminates the basis of the diseases linked to mutations fusion components. Finally, by probing the mechanistic link between mitochondrial fusion and apoptosis, our experiments will provide fundamental information regarding the function and regulation of the Bcl-2 family of proteins, which will impact how scientists view the regulation of apoptosis.